Advancing chemical mixture toxicity assessment using advanced in vitro NAMs: current status and future perspectives

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

• Level A · Stronger Clinical Evidence
• Level B · Emerging clinical evidence with positive signals
• Level C · Early human research exploring benefits
• Level D · Scientific groundwork from lab and animal studies
• Emerging · Emerging topic under active research

Abstract (original English)

Chemical exposure in real-world settings occurs predominantly as mixtures, whereas toxicological assessment has long been shaped by single-substance paradigms. As interest in more human-relevant and mechanistically informative testing strategies has grown, new approach methodologies (NAMs) have been increasingly incorporated into mixture toxicity research. However, the role of advanced in vitro NAMs in chemical mixture toxicity assessment has not yet been examined in a structured manner. This review therefore evaluated how advanced in vitro NAMs are currently being applied in mixture toxicity assessment and considered their potential value for regulatory translation. A PubMed-based literature search covering studies published between 2021 and 2025 identified 353 in vitro NAM-based studies related to chemical mixture toxicity, of which 70 were classified as advanced in vitro NAM-based studies and included for in-depth analysis. Among the advanced in vitro NAM categories, route-of-exposure-relevant models were the most frequently applied, followed by Omics, 3D models, stem cell-based models, and HTS/HCI approaches. The reviewed studies addressed diverse mixtures, including air pollutants, PFAS, phthalates, pesticides/herbicides, consumer product-related mixtures, and other environmentally relevant chemical combinations. Across these studies, advanced in vitro NAMs were used not o

Evidence level

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

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